Experimental Study Of Insulin And Chondroitinase ABC Combined Treatment For Acute Injuried Spinal Cord Of Rat

Objective:To study the effect of strategy combined insulin and chond- roitinase ABC on the regeneration and functional recovery of acute injuried spinal cord and discuss its potential mechanisms.Methods: A total of 124 adult male and female Sprague-Dawley(SD) rats (220±50g) were used in these studies. ASCI was performed with a weight-drop device and advanced Allen's method.Six rat groups were rand- omly as follows:Group A(normal,n=4) ,Group B(control, saline treatment), Group C (ChABC treatment), Group D (insulin treatment), Group D (insulin+ChABC treatment) and Group E (Methylprednisolone, MP, treat- ment). B,C,D,E,F group was sub-divided for postinjury 1-day, 3-day, 1-week, 2-week, 4-week and 8-week, each subgroup has 4 rats.Postinjury, PE-0402 tube (0.4mm in outside diameter, 0.2mm in inside diameter) was placed into cavitas subarachnoidealis of Group C and Group E.A 16 gauge indwelling needle was insert into vena caudalis and flushed with 1ml sodium heparin for mice in group E.Drug Treatment:After ASCI 30min, rats were received diverse treatments. Normal rats(group A) were not any intervention.Control rats (group B) were intraperitoneal injection with 1.0ml saline everyday for 1 week. For rats in group C, 6μl (1U/ml) ChABC were first injected and then 6μl saline through the tube for 1 week. For rats in group D,intraperitoneal injection with 1 IU/kg/d insulin with 1 g/kg/d 50% glucose three times a day for 1 week. For rats in group E, insulin and ChABC in combination were injected by their respective ways and times. For rats in group F, MP was injection in vena caudalis through indwelling needle with 30mg/kg for the first time, interval 45min, then 5.4 mg/kg/h for 23hs. Observation index and collection data: Locomotor behavior was monitored and scored according to the Basso, Beattie and Bresnahan (BBB) open field test.Locomotor behavior was tested at postinjury 1 day,3 days,1 week,2 weeks,4weeks,8 weeks.Neuroelectrophysiological Study:All studies were performed by the same skill technician in neuroelectrophysiology. The rats in each group were measured at postinjury 1 day,3 days,1 week,2 weeks,4weeks,8 weeks.Spinal cord evoked potential (SCEP) and motor evoked potential (MEP) were measured. Record the latency and amplitude of SCEP and MEP.After neuroelectrophysiological study, all animals were trans-heart perfused with 4% paraformaldehyde (PFA). Then the spinal cord with spinal column (T8-T10) was cut and postfixed with 4% PFA. Twelve hours later, only spinal cord tissue was drew out and fixed overnight and embedded in 10% gelatin for histomorphology observation. Include HE and Nissl stain, Immunochemistry analysis:apoptosis, GAP-43, BDA tracing.All images were photographed by using SimPCI software, and then collected and analyzed. Morphometric data were analyzed using either the t-test or the one-way ANOVA with Tukey's HSD for postinjury comparisons. Locomotor behavior data were analyzed using the non-Para- metric, Mann-Whitney test. Significance levels were considered at p < 0.05.Results: BBB scores of preinjury is 21,After ASCI , all rats were instantly observed typical paraplegia syndrome, exhibiting a series of symptoms such as paralysis in two hind legs, hypomyotonia.Twelve hours later, rats could start to scramble with two hind legs towing and acrotarsium on the ground. BBB scores of all rats is 0 at postinjury 1 day,3days.BBB scores of rats treated with insulin or insulin+ ChABC or MP were much higher than that of rats treated with control or ChABC, and the peak time of score was at postinjury 2-week. Moreover, the activity of hind-leg rats in insulin or insulin+ChABC or MP treatment groups independently were much power than rats in control or ChABC treatment groups respectively. At postinjury 8-week, the average BBB scores of rats in insulin or insulin + ChABC or MP treatment groups were above 18, while less than 15 in other two groups. And the locomotor ability was much stronger than that of the control or ChABC groups respectively.The latency time and amplitude of SCEP was no significantly different among every group at postinjury 1 day,3days,1 week,4 weeks,8 weeks.The latency time and amplitude of MEP in proximal end of injury could not been detected in the first postinjury week. From the second week postinjury , the latency time and amplitude of MEP in proximal end of injury was gradually recovered in the rats treated with insulin or insulin + ChABC or MP, while that of control- and ChABC-treated rats could not be measured. From postinjury 4- to 8-week, the recovery was much obvious in these three treatment groups. The latency time and amplitude of MEP in both proximal and external end of spinal cord could be detected, and that of external end was more obvious, exhibiting shorter latency time and higher amplitude in rats treated with insulin or insulin + ChABC or MP than that of rats treated with control or ChABC individually.HE staining and Nissl staining: spinal cord extensive hemorrhage, red blood cells exosmotic, neuron nuclear condensed, necrosis and dissolved, microgliasis and inflammatory infiltration, capsular space formation. But in rats treated with insulin or insulin + ChABC or MP better than that of rats treated with control or ChABC individually.The positive GAP-43 cells were appeared at postinjury 1-day, which was mainly expressed in the grey substance, fewer in the white substance. The GAP-43 expression was peak at postinjury 7-day and then decreased. Only few cells expressed GAP-43 in spinal cord tissues at postinjury 4-week. At postinjury 7-day, the positive expression of GAP-43 of spinal cord tissues in rats treated with insulin or insulin+ ChABC or MP were much more numbers and deeper staining than in rats treated with control or ChABC individually. The positive apoptotic cells were observed in rat spinal cord tissues at postinjury 1-day, both in grey and white substances. The positive apoptotic cells were increased with days, and attained the positive peak at postinjury 7-day and decreased from then. At postinjury 7-day, the positive apoptotic cells of spinal cord tissues in rats treated with insulin or insulin + ChABC or MP were much less than in rats treated with control or ChABC. The apoptotic cells in rats of all groups were less positive at postinjury 4-week, and almost could not be detected at postinjury 8-week.The regenerated neural axons were found to pass the vesicle cavity in rats C,D,E,F group, while no neural fibers passed in B group.Conclusions:1.Insulin have important effective in growth and development of neurologic, control neuron formation, growth, development, differentiation, prevent neuron apoptosis and promote neuron prolification, could recover the locomotor and sensor function of spinal cord.2.Main mechanism of action is insulin conjugate insulin receptor, activate protein tyrosine kinase(PTK) of insulin receptorβsubunit, PTK effect its substrate, cause signal transduction cascade reaction magnify,Pri- ming biological effect of insulin prevent apoptosis.The way is IRS-1-Ras- MAPK and IRS-1-PI3K-PKB.3.Chondroitinase ABC can destroy chondroitin sulfate proteoglycans specificity, prevent scarring, profit axon growth, indirect promote injuried spinal cord regeneration and recovery.4.Combined insulin and Chondroitinase ABC can promote the regene- ration of injuried spinal cord and the recovery of hind limb function more evidently than other individually treatment groups.The effect equivalency MP.It could be effective therapeutic ways and leads a potential way for acute traumatic spinal cord injury.